Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by cytopenia and a propensity to develop acute myeloid leukemia (AML). The management of lower-risk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag (EPAG), a thrombopoietin receptor agonist, can improve platelet counts in LR-MDS and tri-lineage hematopoiesis in aplastic anemia (AA). We conducted a phase 2 dose modification study to investigate the safety and efficacy of EPAG in LR-MDS. EPAG dose was escalated from 50 mg/day, to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a PNH clone, marrow hypocellularity, thrombocytopenia with or without other cytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with previous EPAG studies in AA; no patients discontinued drug due to adverse events. Three patients developed reversible grade-3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued EPAG after achieving a robust response (median time 16 months); four of them reinitiated EPAG due to declining counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to AML on study. In conclusion, EPAG was well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.

Purpose The acquisition of pathogenic variants in the TERT promoter ( TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes. Methods We screened patients with a broad spectrum of telomeropathies ( n = 136), their relatives ( n = 52), and controls ( n = 195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay. Results Pathogenic −124 and −146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging. Conclusion We extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.

Background: Immune aplastic anemia (SAA) disproportionally affects children and young adults. Immunosuppression (IST) remains the treatment of choice for patients less than 40 years of age without a fully human leukocyte antigen (HLA) matched sibling. In the pediatric population (aged <18 years), from NIH historic studies, horse antithymocyte globulin (h-ATG) and cyclosporine(CSA) regimes have produced good results, with an overall response rate (ORR) of 74% at 6 months, a relapse rate of 33% at 10 years, and an overall survival (OS) of 80% at 10 years (Scheinberg P, J Pediatrics 2008), generally better outcomes than in adults. Eltrombopag (EPAG) is an oral thrombopoietin-receptor agonist that has been shown to significantly improve response rates when combined with h-ATG and CSA for treatment-naïve patients (Townsley D, NEJM 2017). We report here a pediatric subgroup analysis of our clinical trial (NCT01623167) to assess whether the addition of EPAG to IST improved ORR in children with SAA. Methods: Between 2012 and 2018 a total of 39 patients <18 years of age with SAA were treated with front line EPAG in combination with h-ATG and CSA. We assessed ORR at 6 months, relapse rate, rate of clonal evolution, and OS. Additionally, we monitored for the development of clonal evolution and for the emergence of somatic clones. Germline testing for inherited marrow failure was performed in all patients. Response was defined by blood counts no longer satisfying criteria for SAA. Outcomes were compared to two treatment-naive cohorts: adult patients aged 18 years and older who received EPAG in combination with immunosuppression (adult EPAG group), and a historical cohort (from 1989 to 2010) of 87 pediatric patients aged <18 years who were treated with immunosuppression (pediatric IST group). The pediatric IST group included all patients treated with a h-ATG based regimen including h-ATG with CSA, and h-ATG with CSA and additional immunosuppression (either mycophenolate mofetil or sirolimus). A historical adult group (Adult IST group) of 322 patients treated from 1989 to 2010 with the same IST regimens as the pediatric IST group was also used as a comparator. Analyses were based on intention-to-treat. Results: Median age in the pediatric EPAG group was 15 years and in the pediatric IST group 11 years. Thirty-two patients (of an initial 39) were evaluable for response at 6 months. Seven patients (18%) were taken off study prior to 6 months to undertake bone marrow transplant, due to either failure to respond by 3 months or clinical urgency in count recovery. Overall, 28 (72%) of all treated patients achieved a response at 6 months, by comparison, responses were observed in 74% of the pediatric IST group (p=0.836) and in 83% in the adult EPAG group (p=0.143). In contrast, patients in the adult EPAG group had a significant difference in 6 month ORR compared with the adult IST group (83% versus 58%, p= <0.0001). The PK analysis of EPAG suggested comparable exposure in pediatric (6-17 years) and adult population. Of the 28 patients who achieved a response at 6 months, 43% relapsed, compared to 28% in the pediatric IST group (p=0.252). Median time to relapse in the pediatric EPAG group was 565 days from IST. There was no significant difference in survival between the pediatric EPAG group and either the pediatric IST group or the adult EPAG group. Clonal evolution occurred in 3 (8%) of patients (monosomy 7, del 5q, and translocation 5:12); 7% of patients evolved in the pediatric IST group. Somatic mutation testing was performed in 36 patients at baseline and was negative in all. At the 6 month timepoint, 5 patients had developed new somatic mutations (2 ASXL1, 2 ATRX, 1 BCOR), three clones appeared in responders and two in non-responders. Germline testing did not reveal any pathogenic variants. Conclusion: Eltrombopag added to immunosuppression did not improve ORR at 6 months in pediatric patients with SAA compared to our historical cohort of pediatric IST patients. In contrast, adults had much improved responses with EPAG. Some patients were taken off study prior to 6 month evaluation (18% compared to 6% in the pediatric IST group) to pursue BMT, likely reflecting recent improvements in outcomes and donor availability. In our study the addition of eltrombopag to IST did not provide any obvious therapeutic benefit to pediatric patients with severe aplastic anemia. This research was supported in part by the Intramural Research Program of the NIH and NHLBI. Disclosures No relevant conflicts of interest to declare.

Telomerase reactivation by acquisition of mutations in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. The most common TERTp mutations are located in positions -146, -124, and -57 upstream the initiation codon. In non-malignant diseases, TERTp mutations only have been reported in patients with idiopathic pulmonary fibrosis (IPF) caused by germline mutations in telomere biology genes, that are also etiologic in a broader spectrum of diseases collectively named telomeropathies (such as IPF, aplastic anemia [AA], dyskeratosis congenita [DC], and cirrhosis). We screened blood from 136 patients with telomeropathies (median age=29 years; range, 1-76), 52 relatives (median age=40 years; range, 8-72), and 195 controls using a customized low-cost amplicon-based next-generation sequencing (NGS) assay for identification and quantification TERTp mutations. Patients had DC (n=21), AA (n=86), IPF with or without another telomeropathy-related phenotype (n=18), or other phenotypes (n=11). Inclusion criteria were telomere length (TL) below the 10th percentile of age-matched controls or a germline mutation in a telomere-related gene classified as pathogenic/likely pathogenic or of uncertain significance by the ACMG criteria. Patients' relatives were only studied if they carried the same germline mutation as the proband or had short telomeres, regardless of symptoms or evidence of disease. Patients with acquired AA (n=70), IPF (n=12), other inherited bone marrow failure (n=7), and acute myeloid leukemia (AML; n=106) were controls. All TERTp mutations identified by NGS were confirmed and tracked over time by droplet digital PCR. We identified the -124 or -146 mutations in leukocytes from 12 unrelated patients diagnosed with IPF (n=6), DC (n=2), or moderate AA (n=4). Five relatives also had the -146 (n=1), and -124 (n=4) mutations, all carriers of a germline mutation in telomere biology gene. The frequency of TERTp mutations was much higher in IPF patients compared to AA cases (33% vs. 4.6%; Fisher's exact test, P=0.0016). However, no difference in frequency of TERTp mutations among patients with IPF vs. marrow failure was observed (41% vs. 58%; Fisher's exact test, P>0.05), suggesting TERTp mutations occurred in both clinical presentations. MutTERTp clones positively correlated with age, as they were only present in individuals older than 18 years old and more frequent in those 60 to 80 years old. Also, TERTp mutations more frequently co-ocurred with germline TERT mutations (n=13) compared to mutations in TERC (n=2), RTEL1 (n=1), or DKC1 (n=2) (76% vs. 23%; Fisher's exact test, P=0.002). All germline variants were pathogenic or had some evidence of pathogenicity. MutTERTp clones size varied from 1.2% to 50% in total leukocytes and was at higher allele frequencies (VAF) in the granulocytic fraction from four patients. In serial samples (available for five patients), the mutTERTp clone size expanded over time, suggesting a selective growth advantage in comparison to unmutated hematopoietic cells. Despite that, mutTERTp clones did not associate with blood counts or telomere elongation; most subjects carrying a TERTp mutation, which is known to upregulate TERT expression, nevertheless had short or very short telomeres (15 out of 17 individuals). Six patients with mutTERTp clones (VAF ranging from 3-33% in myeloid cells) were treated with danazol for two years; four were responders and two were off-study after 3-6 months. In serial samples (available for two patients), the mutTERTp clone sizes decreased during danazol treatment while blood counts improved. After treatment, mutTERTp clones VAF increased. TERTp mutations were found in telomeropathy patients who had a germline variant in telomere biology genes but not in controls or patients with very short telomeres without germline variant in telomere biology genes. We have expanded the spectrum of non-malignant diseases associated with somatic TERTp mutations to DC, AA, and cirrhosis. Our data indicate that mutTERTp clones are specifically and randomly selected with aging in a marrow environment deficient in telomerase function, and mutTERTp selection did not associate with patients' peripheral blood counts, TL, and response to danazol treatment. TERTp emergence may be a useful clonal indicator for telomere dysfunction and may help to assess the pathogenicity of unclear constitutional variants in telomeropathies. Disclosures Young: CRADA with Novartis: Research Funding; National Institute of Health: Research Funding; GlaxoSmithKline: Research Funding.

No abstract

No abstract

Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been shown to improve hematopoiesis in patients with aplastic anemia (AA), but in MDS patients the effect of thrombopoietin mimetics in bone marrow function is still unclear. In this phase-2 dose escalation study, we investigated the safety and effectiveness of EPAG treatment in low to intermediate-2 risk MDS patients (NCT 00961064). Thirty patients were enrolled from March 2011 to July 2017. Preceding enrollment the majority of patients were either diagnosed with AA (n=13) or hypoplastic MDS (n=5). EPAG was started at 50 mg/day, up to a maximal dose of 150 mg/day, increasing the dose by 25mg every 2 weeks. The primary endpoint was hematologic response at 16 or 20 weeks, defined as either: (1) an increase in platelet counts ≥20.000/uL or transfusion independence for a minimum of 8 weeks; (2) hemoglobin (Hb) increase of ≥1.5g/dL from baseline, or a reduction in red blood cells (RBC) transfusion of at least 50%; or (3) an increase in absolute neutrophil counts (ANC) of ≥0.5x109/L or by at least 100% in patients with a baseline ANC <0.5x109/L. Responding patients could continue EPAG treatment on an extension arm. The primary endpoint of hematological response was met in 14/30 patients (47%). All responders continued EPAG on the extension arm. In 3 patients, peripheral blood cell counts declined on EPAG after the initial response. One patient withdrew from the study. Ten of the 14 responding patients achieved a robust response (RR) after a median treatment duration of 15 months (range 7-27 months). Robust response was defined as stable hematopoiesis with at least a hemoglobin >10g/dl, and thrombocytes >50.000/L, and ANC>1000/L. However, peripheral blood cell counts significantly declined in 5/10 RR and EPAG was restarted per protocol. In 4 of these patients peripheral blood cell counts recovered. One patient did not achieve a second response. Based on International Prognostic Score System (IPSS), 4/30 (13%) patients progressed on study, including 3 non-responders and 1 responder, at a median follow-up of 4 months (3-35 months). The responding patient was diagnosed with increased bone marrow myeloblast 7 months after discontinuation of EPAG for robust response and 35 months after enrolling in the study. New cytogenetic abnormalities determined progression in non-responding patients (Figure). Novel dose limiting toxicities were not observed. Three patients developed CTCAE grade III hepatic toxicities. One of them discontinued EPAG at 3 months. Elevated transaminases returned to baseline after EPAG discontinuation in 2 patients. In both cases EPAG was resumed either at the same (150mg/day) or reduced dose (50mg/day) level. There were no treatment-related death cases. One patient died on study before the primary endpoint from acute respiratory distress syndrome. Sequential acquisition of genomic aberrations has been associated with malignant transformation. Targeting next-generation sequencing for somatic variants in genes previously associated with myeloid malignancies (Myeloid cancer genes, MCG) was performed in 29/30 patients with sufficient material (bone marrow mononuclear cells) available from baseline, primary endpoint, and at time of progression. At baseline, 22/29 (76%) patients were found with at least one mutation:TET2 (14.5%), ASXL1 (12.5%), SF3B1 (8.3%), SETBP1 (8.3%), ATM (8.3%), and ZRSR2 (8.3%). After EPAG, additional somatic variants in different genes were detected in 4/14 responders and 7/16 non-responders. Variants present at baseline were no longer detected in post EPAG samples from 4 responding and 6 non-responding patients. The VAF of variants detected at both time points were similar, indicating no selective expansion of clones with EPAG in neither responder, non-responder nor patients with progression based on IPSS. In conclusion, our results suggest that EPAG is well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-2 risk MDS, particular with a prior history of hypoplastic bone marrow failure syndromes. EPAG was discontinued for robust response in the majority of responders but declining blood cell counts were observed in about 50% of them. Variants in MCG were more common at study entry compared to patients with aplastic anemia (Yoshizato, NEJM, 2015). However, EPAG appears not to selectively promote expansion of clones harboring MCGs in this patient population. Disclosures Townsley: National Institute of Health: Research Funding. Scheinberg:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding. Dunbar:National Institute of Health: Research Funding. Young:GlaxoSmithKline: Research Funding; CRADA with Novartis: Research Funding; National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.